Comparison of DNA extraction methods to detect Pseudogymnoascus destructans in bat skin swabs,guano,and environmental swabs

Abstract

National surveillance for Pseudogymnoascus destructans—the fungus that causes white-nose syndrome in bats—relies on methods optimized for highly sensitive detection of the pathogen. However, comparisons of methods that inform assay functionality are sparse. We used a combination of conidia-spiking experiments and analysis of field-collected samples to compare methods for DNA extraction from bat guano, skin swabs, and environmental swabs for subsequent detection of P. destructans DNA via quantitative real-time PCR (qPCR). We evaluated 2 methods for all sample types and a third method for guano samples. We created conidia-inoculated guano samples from 3 guano sources, as well as conidia-inoculated environmental swabs using sediment from 7 bat hibernacula. Detection of P. destructans from environmental swabs was 2.94 times more likely from a purification-free method of DNA extraction, with up to 9.75 times more target DNA detected on average than a magnetic bead purification method, as evidenced by both laboratory-spiked and field-collected samples. We observed a similar result for skin swabs collected from P. destructans–infected bat carcasses, with a 31.7-fold increase in the amount of target DNA detected on average. Between the same 2 extraction methods, a 3.3-fold difference was found in the odds of detecting P. destructans from guano, but a third method designed for DNA extraction from soil had 12.6–41.8 times higher odds of detection. Our study further optimizes DNA extraction methods for the detection of P. destructans from environmental swabs and offers a detailed comparison of extraction methods used for detection from bat skin swabs and guano.